The goals of this project are to delineate the roles of the polymerase activity and the 3' to 5' exonuclease activity of DNA polymerase I in maintaining the fidelity of DNA synthesis, and to elucidate the mechanisms of template-directed polymerization and proof-reading at the molecular level. The molecular mechanisms of template-directed polymerization and proof-reading are being investigated by inhibitor studies, by studies on the relative effects of the hydrogen-bonding properties of nucleotides at the primer terminus on polymerization and hydrolysis of nucleotides, and by studies on the effects of enol-keto tautomerization of bases on the fidelity of DNA synthesis. The data suggests that the active sites for the polymerase and 3' to 5' exonuclease activities of DNA polymerase I are seperate and distinct and that the primer terminus of the primer/template, a substrate for both activities, can bind to either active site. The binding of a primer terminus at the polymerase site is restricted to termini that are able to base-pair with the template strand and is dictated by the base sequence of the template. In contrast, binding of a primer terminus at the exonuclease site requires that the terminus be unpaired and is, therefor, favored for termini that are less able to basepair with the template.